1. Field of the Invention
It is known that when a heavy atom is able to contact a fluorescer, quenching results. This phenomenon can be employed in any assay, where a ligand or ligand analog of interest naturally has a heavy atom, such as iodine, or the heavy atom is synthetically introduced. By preparing a conjugate of the heavy atom containing ligand and a fluorescer, a relatively low level of fluorescence is observed, when the conjugate is irradiated with light at a wavelength which results in excitation of the fluorescer. However, when antiligand is bound to the ligand in the conjugate, a substantial enhancement of fluorescence is observed.
With polyiodothyronines, the iodine present is capable of quenching fluorescence, hen the polyiodothyronine is covalently bonded to a fluorescer. However, when attempting to use this reagent in a serum sample for determining polyiodothyronines, non-specific binding of serum proteins to the conjugate results in variation in the observed fluorescence unrelated to the amount of ligand present. Due to patient sample variation, the degree to which the observed fluorescence changes at constant ligand concentration varies with the source of the serum. Therefore, it is necessary to find some means to inhibit the non-specific effect of the serum proteins on the observed results.
2. Description of the Prior Art
U.S. Pat. No. 3,988,943 describes a competitive protein binding assay employing ligand-fluorescer conjugates, where the binding of antiligand inhibits the binding of antifluorescer. U.S. Pat. No. 3,996,345 describes an immunoassay employing a chromophore pair, where the chromophores are related by one of the chromophores quenching the fluorescence of the other one of the chromophores, where the amount of quencher brought within quenching distance of the fluorescing chromophore is related to the amount of analyte in the sample. Robbins, "Thyroxine-binding Proteins", Trace Components of Plasma: Isolation and Clinical Significance, Alan, R. Liss, Inc., New York, page 331 (1976) postulated that the inability of prealbumin to bind thyroxine-agarose affinity gels was related to the inability of the thyroxine to orient properly in the protein binding site. Co-pending application Ser. No. 824,576, filed Aug. 13, 1977, teaches the use of a polyiodothyronine-fluorescer conjugate for the determination of polyiodothyronines based on the enhancement of fluorescence when anti(polyiodothyronine) binds to the polyiodothyronine.